To define basic mechanisms involved in type II cell-specific, developmental and hormonal regulation of surfactant synthesis in fetal lung we have focused on surfactant protein-A (SP-A), which is expressed in lung alveolar type II cells and is developmentally regulated in concert with surfactant phospholipid synthesis. We found that the fetal baboon serves as an excellent model for the human with regard to the surfactant protein-A (SP- A) genes and their regulation. Recently, we found that the transcription factor (TF) TTF-1 plays an essential role in cAMP induction of SP-A promoter activity. cAMP treatment of type II cells increases TTF-1 phosphorylation, DNA-binding and transcriptional activity. Developmental and cAMP-induced changes in TTF-1 phosphorylation and DNA-binding may facilitate its interaction with other TFs and co-activators, resulting in an induction of SP-A promoter activity. To identify factors that interact with TTF-1, we will use a novel a yeast two-hybrid system. cDNAs of interest will be analyzed to study developmental regulation, cell-specific expression, as well as their ability to activate the SP-A promoter. We also have observed that the inductive effect of cAMP on SP-A gene expression is dependent upon a critical level of environmental O2. To define mechanisms for the permissive role of O2 on cAMP induction of SP-A gene expression, we will analyze effects of environmental O2 on binding of fetal lung nuclear proteins to previously characterized response elements within the 5'-flanking region of the bSP-A gene. Changes in the levels of the O2-regulated TFs NF-kappaB, its inhibitory partner IkappaB, HIF-1alpha and the related protein EPAS1/HLF in lung tissues of fetal baboons during development and after organ culture in 2 percent vs. 20 percent O2 will be analyzed. Previously, we found that lipogenic TFs C/EBPdelta, PPARgamma and SREBP-1 and -2 are expressed in type II cells and induced with type II cell differentiation. Expression, cellular/subcellular localization and DNA-binding activity of these TFs will be analyzed in baboon fetal lung during development and in association with changes in surfactant synthesis. We also have observed that postnatal ventilation of prematurely delivered baboons (125 days) resulted in a 4-fold increase in DSPC pool sizes to levels similar to those of term animals; postnatal Dex treatment stimulated increased mobilization of DSPC and SP-B into the airways without having an effect on total tissue pool sizes. The marked induction of surfactant phospholipid synthesis in the transition to air-breathing suggests that dramatic changes in O2 availability to the type II cell may play an important role in the induction of surfactant synthesis. Effects of ventilation and systemic treatment with Dex and Bt2cAMP on surfactant protein and phospholipid synthesis will be correlated with expression of the O2-regulated and lipogenic TFs. The studies outlined in this proposal should advance our understanding of basic mechanisms involved in developmental and hormonal regulation of type II cell differentiation and surfactant synthesis and facilitate development of therapies to accelerate lung development and surfactant production in prematurely born infants to avoid the deleterious effects of mechanical ventilation resulting in BPD.